Methods for telomere length and genomic dna quality control analysis in pluripotent stem cells

Abstract

The generation of clinical-grade cell-based therapies from human embryonic stein cells or cells reprogrammed to pluripotency from somatic cells, requires stringent quality controls to insure that the cells have long enough telomeres and resulting cellular lifespan to be clinically useful, and normal gene expression and genomic integrity so as to insure cells with a desired and reproducible phenotype and to reduce the risk of the malignant transformation of cells. Assays useful in identifying human embryonic stem cell lines and pluripotent cells resulting from the transcriptional reprogramming of somatic cells that have embryonic telomere length are described as well as quality control assays for screening genomic integrity in cells expanded and banked for therapeutic use, as well as assays to identify cells capable of abnormal immortalization.

1 .- 14 . (canceled) 
     
     
         15 . An induced pluripotent stem cell (iPS) identified by evaluating telomere length of a pluripotent stem cell line, wherein said iPS is suitable for clinical use if said telomere length is restored to near-embryonic telomere length. 
     
     
         16 . The iPS cell of  claim 15 , wherein said pluripotent cell line displays a normal karyotype. 
     
     
         17 . A system for identifying a pluripotent stem cell capable of restoring telomere length comprising:
 a gene expression level evaluation element configured for evaluating the level of expression of at least one gene in a pluripotent stem cell to obtain a gene expression level result, wherein the at least one gene is selected from one or more of: PCNA, CDC2, MSH2, ZNF146, TERF1 transcript variant 2, VENTX and PRKDC; and   a phenotype determination element configured for employing the gene expression level result to identify a pluripotent stem cell capable of restoring telomere length.   
     
     
         18 . The system of  claim 17 , wherein the phenotype determination element comprises a reference gene expression level result. 
     
     
         19 . The system of  claim 18 , wherein the reference gene expression level result is from embryonic stem cells. 
     
     
         20 . The system of  claim 19 , wherein the embryonic stem cells are human embryonic stem cells with telomere restriction fragment lengths of 12 to 18 kilobases (kb). 
     
     
         21 . The iPS of  claim 15  comprising exogenous VENTX, wherein the exogenous VENTX is VENTX protein or a nucleic acid encoding for VENTX protein. 
     
     
         22 . The iPS of  claim 15 , wherein the expression level of one or more genes selected from PCNA, CDC2, MSH2, ZNF146, TERF1 transcript variant 2, VENTX and PRKDC is comparable to the expression level of said gene in a reference human embryonic stem (hES) cell line.